HIV-1 transmission via breastfeeding remains a major mode of infant HIV-1 acquisition in areas of high HIV prevalence, accounting for nearly half of the 350,000 new infant HIV-1 infections occurring annually. Maintaining the immunologic and nutritional benefits of breastfeeding, while protecting the infant against HIV-1 acquisition, is critical for HIV-free survival of infants in resource-poor regions of high HIV-1 prevalence. While antiretroviral drug prophylaxis administered to the infant and/or mother reduces postnatal HIV-1 transmission, concerns of toxicities and development of antiretroviral drug-resistant strains limit the success of this strategy. Therefore, novel strategies to prevent postnatal HIV-1 transmission via breastfeeding will be necessary to eliminate this mode of infant HIV-1 acquisition. Interestingly, in the absence of maternal/infant antiretroviral prophylaxis, only 10% of breastfeeding infants born to HIV-1-infected mothers will acquire HIV-1 via breastfeeding, despite chronic, daily HIV-1 exposure. This low rate of transmission suggests that innate factors in breast milk protect the majority of infants against virus acquisition. Through detailed fractionation and screening of proteins in breast milk for HIV-neutralizing activity, we have isolated a novel, potent HIV- neutralizing protein from breast milk. We hypothesize that this protein is responsible for protection of the overwhelming majority of HIV-exposed breastfeeding infants against HIV-1 acquisition. In this proposal, we will explore the interactions of this novel HIV-neutralizing protein with the HIV-1 Envelope, defining its active site and the neutralizing epitope of the HIV-1 Envelope that mediates the virus inhibition, potentially uncovering a unique target for HIV-1 neutralization. Furthermore, we will investigate the in vivo efficacy of this protin by investigating the association between the endogenous concentration in milk and protection against postnatal HIV-1 transmission in a large cohort of HIV-infected mother-infant pairs. This work will define the mechanism and efficacy of this newly-identified HIV-1-neutralizing protein isolated from breast milk, establishing a novel, safe prophylactic agent to protect infants against HIV-1 acquisition via breastfeeding. This proposal is being submitted in response to the NIAID Program Announcement PA-11-217 Mechanistic Studies of HIV-exposed Seronegative Individuals, specifically addressing innate factors that naturally protect the majority of highly HIV-exposed, breastfeeding infants against postnatal HIV-1 acquisition.